Use Of Toll-like Receptors Agonists Research Articles

Recent Insights into the Molecular Mechanisms of the Toll-like Receptor Response to Influenza Virus Infection.

Influenza A viruses (IAVs) pose a significant global threat to human health. A tightly controlled host immune response is critical to avoid any detrimental effects of IAV infection. It is critical to investigate the association between the response of Toll-like receptors (TLRs) and influenza virus. Because TLRs may act as a double-edged sword, a balanced TLR response is critical for the overall benefit of the host. Consequently, a thorough understanding of the TLR response is essential for targeting TLRs as a novel therapeutic and prophylactic intervention. To date, a limited number of studies have assessed TLR and IAV interactions. Therefore, further research on TLR interactions in IAV infection should be conducted to determine their role in host-virus interactions in disease causation or clearance of the virus. Although influenza virus vaccines are available, they have limited efficacy, which should be enhanced to improve their efficacy. In this study, we discuss the current status of our understanding of the TLR response in IAV infection and the strategies adopted by IAVs to avoid TLR-mediated immune surveillance, which may help in devising new therapeutic or preventive strategies. Furthermore, recent advances in the use of TLR agonists as vaccine adjuvants to enhance influenza vaccine efficacy are discussed.

TLR Agonists Delivered by Plant Virus and Bacteriophage Nanoparticles for Cancer Immunotherapy.

Toll-like receptors (TLRs) are promising targets in cancer immunotherapy due to their role in activating the immune system; therefore, various small-molecule TLR agonists have been tested in clinical applications. However, the clinical use of TLR agonists is hindered by their non-specific side effects and poor pharmacokinetics. To overcome these limitations, we used plant virus nanoparticles (VNPs) and bacteriophage virus-like particles (VLPs) as drug delivery systems. We conjugated TLR3 or TLR7 agonists to cowpea mosaic virus (CPMV) VNPs, cowpea chlorotic mottle virus (CCMV) VNPs, and bacteriophage Qβ VLPs. The conjugation of TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), resulted in the potent activation of immune cells and promoted the production of pro-inflammatory cytokine interleukin 6. We found that 1V209 conjugated to CPMV, CCMV, and Qβ reduced tumor growth in vivo and prolonged the survival of mice compared to those treated with free 1V209 or a simple admixture of 1V209 and viral particles. Nucleic acid-based TLR3 agonist, polyinosinic acid with polycytidylic acid (poly(I:C)), was also delivered by CPMV VNPs, resulting in enhanced mice survival. All our data suggest that coupling and co-delivery are required to enhance the anti-tumor efficacy of TLR agonists and simple mixing of the VLPs with the agonists does not confer a survival benefit. The delivery of 1V209 or poly(I:C) conjugated to VNPs/VLPs probably enhances their efficacy due to the multivalent presentation, prolongation of tumor residence time, and targeting of the innate immune cells mediated by the VNP/VLP carrier.

Differential Effects of Toll-Like Receptor Signaling on the Activation of Immune Responses in the Upper Respiratory Tract.

ABSTRACTVaccination through the upper respiratory tract (URT) is highly effective for the prevention of respiratory infectious diseases. Toll-like receptor (TLR)-based adjuvants are immunostimulatory and considered potential adjuvant candidates. However, the patterns of immune response to different TLRs at the URT have not been revealed. In this study, SPF mice were preexposed to TLR agonists intranasally to simulate the status of humans. Inflammatory response to TLR agonists and TLR signal-mediated adaptive immune responses were analyzed. The results revealed that similar to human tonsils, inflammatory response to stimulation with TLR4 or TLR2 agonist was attenuated in agonist-exposed mice but not in mice without this exposure. In contrast, TLR9 or TLR3 agonist preexposure did not affect the inflammatory response to restimulation by matching agonists. For the adaptive immune response, after agonist preexposure the antibody response to antigens adjuvanted with TLR4 or TLR2 agonist was substantially restricted, whereas, both antibody and T cell responses to antigens adjuvanted with TLR9 or TLR3 agonist were activated as robustly as in mice without agonist exposure. Moreover, we demonstrate that the mechanisms underlying the differential activation of TLRs are regulated at the level of TLR expression in innate and adaptive immune cells. These results indicate that TLRs on the cell surface (TLR4 and 2) and in the endolysosomal compartments (TLR9 and 3) display distinct immune response patterns. The findings provide important information for the use of TLR agonists as mucosal adjuvants and enhance our understanding of immune responses to bacterial and viral infections in the respiratory mucosa.IMPORTANCE Agonists of TLRs are potential adjuvant candidates for mucosal vaccination. We demonstrated that the TLR-mediated inflammatory and antibody responses in the URT of SPF mice exposed to extracellular TLR agonists were substantially restricted. In contrast, inflammatory and adaptive immune responses, including B and T cell activation, were not desensitized in mice exposed to intracellular TLR agonists. The distinct responsive patterns of extra and intracellular TLRs regulated at TLR expression in immune cells. The results indicated that TLRs differentially impact the innate and adaptive immune response in the URT, which contributes to the selection of TLR-based mucosal adjuvants and helps understand the difference between the immune response in bacterial and viral infections.

An Overview of Recent Insights into the Response of TLR to SARS-CoV-2 Infection and the Potential of TLR Agonists as SARS-CoV-2 Vaccine Adjuvants.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to coronavirus disease (COVID-19), a global health pandemic causing millions of deaths worldwide. However, the immunopathogenesis of COVID-19, particularly the interaction between SARS-CoV-2 and host innate immunity, remains unclear. The innate immune system acts as the first line of host defense, which is critical for the initial detection of invading pathogens and the activation and shaping of adaptive immunity. Toll-like receptors (TLRs) are key sensors of innate immunity that recognize pathogen-associated molecular patterns and activate downstream signaling for pro-inflammatory cytokine and chemokine production. However, TLRs may also act as a double-edged sword, and dysregulated TLR responses may enhance immune-mediated pathology, instead of providing protection. Therefore, a proper understanding of the interaction between TLRs and SARS-CoV-2 is of great importance for devising therapeutic and preventive strategies. The use of TLR agonists as vaccine adjuvants for human disease is a promising approach that could be applied in the investigation of COVID-19 vaccines. In this review, we discuss the recent progress in our understanding of host innate immune responses in SARS-CoV-2 infection, with particular focus on TLR response. In addition, we discuss the use of TLR agonists as vaccine adjuvants in enhancing the efficacy of COVID-19 vaccine.

The Use of Toll-Like Receptor Agonists in HIV-1 Cure Strategies.

Toll-like receptors (TLRs) are a family of pattern recognition receptors and part of the first line of defense against invading microbes. In humans, we know of 10 different TLRs, which are expressed to varying degrees in immune cell subsets. Engaging TLRs through their specific ligands leads to activation of the innate immune system and secondarily priming of the adaptive immune system. Because of these unique properties, TLR agonists have been investigated as immunotherapy in cancer treatment for many years, but in recent years there has also been growing interest in the use of TLR agonists in the context of human immunodeficiency virus type 1 (HIV-1) cure research. The primary obstacle to curing HIV-1 is the presence of a latent viral reservoir in transcriptionally silent immune cells. Due to the very limited transcription of the integrated HIV-1 proviruses, latently infected cells cannot be targeted and cleared by immune effector mechanisms. TLR agonists are very interesting in this context because of their potential dual effects as latency reverting agents (LRAs) and immune modulatory compounds. Here, we review preclinical and clinical data on the impact of TLR stimulation on HIV-1 latency as well as antiviral and HIV-1-specific immunity. We also focus on the promising role of TLR agonists in combination strategies in HIV-1 cure research. Different combinations of TLR agonists and broadly neutralizing antibodies or TLRs agonists as adjuvants in HIV-1 vaccines have shown very encouraging results in non-human primate experiments and these concepts are now moving into clinical testing.

Critical Role of B Cells in Toll-Like Receptor 7-Mediated Protection against Listeria monocytogenes Infection.

Toll-like receptors (TLR) trigger the immune system to mount a rapid innate response capable of protecting the host from a wide variety of bacterial and viral pathogens. There is interest in harnessing TLR agonists to reduce the susceptibility of at-risk populations to infection. However, the widespread prophylactic use of TLR agonists has been compromised by the need to administer them by parenteral injection. An exception is the TLR7/8 agonist R848, which can boost gastrointestinal and systemic immunity when administered orally. This work examines the effect of R848 on host susceptibility to Listeria monocytogenes in a murine challenge model and describes the underlying mechanisms. Results show that prophylactic administration of R848 significantly reduces susceptibility to infection of BALB/c mice, an effect that lasts 1 week. Oral R848 directly stimulated B cells to produce cytokines and Ig. In the absence of B cells, R848-mediated protection was lost. These findings support the use of oral R848 to reduce the susceptibility of at-risk individuals to infection and identify the critical role of B cells in TLR7-mediated resistance to bacterial infection.

Targeting Cellular and Tissue HIV Reservoirs With Toll-Like Receptor Agonists.

The elimination of both cellular and tissue latent reservoirs is a challenge toward a successful HIV cure. “Shock and Kill” are among the therapeutic strategies that have been more extensively studied to target these reservoirs. These strategies are aimed toward the reactivation of the latent reservoir using a latency-reversal agent (LRA) with the subsequent killing of the reactivated cell either by the cytotoxic arm of the immune system, including NK and CD8 T cells, or by viral cytopathic mechanisms. Numerous LRAs are currently being investigated in vitro, ex vivo as well as in vivo for their ability to reactivate and reduce latent reservoirs. Among those, several toll-like receptor (TLR) agonists have been shown to reactivate latent HIV. In humans, there are 10 TLRs that recognize different pathogen-associated molecular patterns. TLRs are present in several cell types, including CD4 T cells, the cell compartment that harbors the majority of the latent reservoir. Besides their ability to reactivate latent HIV, TLR agonists also increase immune activation and promote an antiviral response. These combined properties make TLR agonists unique among the different LRAs characterized to date. Additionally, some of these agonists have shown promise toward finding an HIV cure in animal models. When in combination with broadly neutralizing antibodies, TLR-7 agonists have shown to impact the SIV latent reservoir and delay viral rebound. Moreover, there are FDA-approved TLR agonists that are currently being investigated for cancer therapy and other diseases. All these has prompted clinical trials using TLR agonists either alone or in combination toward HIV eradication approaches. In this review, we provide an extensive characterization of the state-of-the-art of the use of TLR agonists toward HIV eradication strategies and the mechanism behind how TLR agonists target both cellular and tissue HIV reservoirs.

Toll-Like Receptors as Therapeutic Targets in Central Nervous System Tumors.

In recent years, progress has been made in understanding the pathological, genetic, and molecular heterogeneity of central nervous system (CNS) tumors. However, improvements in risk classification, prognosis, and treatment have not been sufficient. Currently, great importance has been placed to the tumor microenvironment and the immune system, which are very important components that influence the establishment and development of tumors. Toll-like receptors (TLRs) are innate immunite system sensors of a wide variety of molecules, such as those associated with microorganisms and danger signals. TLRs are expressed on many cells, including immune cells and nonimmune cells such as neurons and cancer cells. In the tumor microenvironment, activation of TLRs plays dual antitumoral (dendritic cells, cytotoxic T cells, and natural killer cells activation) and protumoral effects (tumor cell proliferation, survival, and resistance to chemotherapy) and constitutes an area of opportunities and challenges in the development of new therapeutic strategies. Several clinical trials have been carried out, and others are currently in process; however, the results obtained to date have been contradictory and have not led to a definitive position about the use of TLR agonists in adjuvant therapy during the treatment of central nervous system (CNS) tumors. In this review, we focus on recent advances in TLR agonists as immunotherapies for treatment of CNS tumors.

Targeting Negative Regulators of TRIF-dependent TLR Signaling Pathway as a Novel Therapeutic Strategy

Background: Toll-Like Receptors (TLRs) are a subclass of pathogen-associated molecular patterns (PAMPs). There is a growing interest in the use of TLR agonists for various pathological dysfunctions, including cancer, microbial infections, and inflammatory diseases. TLR3/4 agonists that can induce TIR-domain-containing adapter-inducing interferon-β (TRIF)- dependent pathway have shown fewer toxic immunostimulatory responses in comparison to other small molecules. Furthermore, TLR3 agonists indicate promising anti-tumor potential in cancer immunotherapy either as vaccine adjuvant or monotherapy. </P><P> Objective: It is logical to assume that the induction of the genes that are involved in TRIF pathway to augment their pleiotropic effects on different cells via TLR agonists, could enhance the treatment process of disease while minimizing the toxicity related to using other small molecules. Methods: An extensive literature search to identify the negative regulators of TRIF-dependent signaling pathway and their biological functions was performed from two databases PubMed and Scopus. Results: Negative regulators of TRIF signaling pathways were identified. In addition, structure and function of sterile α- and armadillo-motif containing protein (SARM), the only TIR domaincontaining adaptor protein that inhibits TRIF-dependent activation, were briefly reviewed. Conclusion: We proposed that the manipulation of TRIF signaling pathway via targeting its negative regulators could be used as an approach to modulate the functions of this pathway without undesired toxic proinflammatory responses.

Abstract P4-06-02: Chemoimmunotherapy with cyclophosphamide plus a toll-like receptor 9 (TLR9) agonist eradicates triple negative breast cancer in a murine model

Abstract Background. Toll-like receptors (TLR) recognize conserved molecular patterns expressed by microbes and together with other molecular sensors serve as a first line of defense, inducing soluble and cellular mediators of innate immunity and initiating key steps of an adaptive immune response. The use of TLR agonists for therapeutic purposes relies on the ability of these compounds to induce, at least partially, some of the immune events that occur during natural infections. For example, synthetic CpG-containing oligodeoxynucleotides (ODN) act as TLR9 agonists, mimicking stimulation of the immune system by bacterial or viral DNA. CpG ODNs are being developed for cancer immunotherapy based on their capacity to stimulate innate and adaptive anti-tumor responses. In this study, we examined the efficacy of intratumorally administered CpG-ODN 1826 alone or in combination with immunomodulatory antibodies (anti-PD-1, anti-OX40, anti-CTLA4) or chemotherapy (cyclophosphamide, paclitaxel) in the syngeneic mouse 4T1 breast tumor model. Methods. Tumors were implanted orthotopically in the mammary fat pads on the left and right flanks while only one tumor was injected with CpG-ODN 1826. Therapy began 12-14 days post tumor injection when tumors were 8-10 mm in diameter. CpG-ODN 1826 (100 ug) was administered intratumorally, while immunomodulatory antibodies (anti-PD-1 [200 ug], anti-CTLA4 [100 ug], anti-OX40 [400 ug]) and chemotherapies (cyclophosphamide [150 mg/kg], paclitaxel [10 mg/kg]) were administered intraperitoneally. Tumor volume was monitored on both flanks to assess direct and abscopal/systemic anti-tumor activity. Tumor tissue obtained during the treatment regimen was used to evaluate therapy-induced changes in the immune microenvironment. Results. CpG-ODN 1826, administered intratumorally over 5 consecutive days induced complete regressions in ˜50% of the treated tumors, but only delayed growth in the distant lesions. The immunomodulatory antibodies had little effect on their own and did not add to the therapeutic efficacy of CpG-ODN 1826. The best therapeutic efficacy was obtained with a combination of weekly cyclophosphamide + CpG-ODN 1826 resulting in complete regression of both the CpG injected tumors and the contralateral tumors in 100% of the mice. Conclusions. These data support the clinical investigation of the combination of a TLR9 agonist (CpG-ODN) with cyclophosphamide in women with breast cancer. Citation Format: Campbell MJ, Tandon V, Senman B, Li J, Esserman L. Chemoimmunotherapy with cyclophosphamide plus a toll-like receptor 9 (TLR9) agonist eradicates triple negative breast cancer in a murine model [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-02.

Targeting Pattern Recognition Receptors (PRR) for Vaccine Adjuvantation: From Synthetic PRR Agonists to the Potential of Defective Interfering Particles of Viruses.

Modern vaccinology has increasingly focused on non-living vaccines, which are more stable than live-attenuated vaccines but often show limited immunogenicity. Immunostimulatory substances, known as adjuvants, are traditionally used to increase the magnitude of protective adaptive immunity in response to a pathogen-associated antigen. Recently developed adjuvants often include substances that stimulate pattern recognition receptors (PRRs), essential components of innate immunity required for the activation of antigen-presenting cells (APCs), which serve as a bridge between innate and adaptive immunity. Nearly all PRRs are potential targets for adjuvants. Given the recent success of toll-like receptor (TLR) agonists in vaccine development, molecules with similar, but additional, immunostimulatory activity, such as defective interfering particles (DIPs) of viruses, represent attractive candidates for vaccine adjuvants. This review outlines some of the recent advances in vaccine development related to the use of TLR agonists, summarizes the current knowledge regarding DIP immunogenicity, and discusses the potential applications of DIPs in vaccine adjuvantation.

Review on Toll-Like Receptor Activation in Myasthenia Gravis: Application to the Development of New Experimental Models.

Abnormal toll-like receptor (TLR) activation and uncontrolled resolution of inflammation are suspected to play a key role in the development of autoimmune diseases. Acquired myasthenia gravis (MG) is an invalidating neuromuscular disease leading to muscle weaknesses. MG is mainly mediated by anti-acetylcholine receptor (AChR) autoantibodies, and thymic hyperplasia characterized by ectopic germinal centers is a common feature in MG. An abnormal expression of certain TLRs is observed in the thymus of MG patients associated with the overexpression of interferon (IFN)-β, the orchestrator of thymic changes in MG. Experimental models have been developed for numerous autoimmune diseases. These models are induced by animal immunization with a purified antigen solubilized in complete Freund's adjuvant (CFA) containing heat-inactivated mycobacterium tuberculosis (MTB). Sensitization against the antigen is mainly due to the activation of TLR signaling pathways by the pathogen motifs displayed by MTB, and attempts have been made to substitute the use of CFA by TLR agonists. AChR emulsified in CFA is used to induce the classical experimental autoimmune MG model (EAMG). However, the TLR4 activator lipopolysaccharide (LPS) has proved to be efficient to replace MTB and induce a sensitization against purified AChR. Poly(I:C), the well-known TLR3 agonist, is also able by itself to induce MG symptoms in mice associated with early thymic changes as observed in human MG. In this review, we discuss the abnormal expression of TLRs in MG patients and we describe the use of TLR agonists to induce EAMG in comparison with other autoimmune experimental models.

Toll-Like Receptor Activation in Immunity vs. Tolerance

After the discovery of Toll-like receptors (TLR) in the late 1990s, initial investigations were focused on understanding their role, stimulating immune responses against infectious agents. Yet, the human body is home to a myriad of TLR agonistic bacteria that have not only established symbiosis with the immune system but also likely contribute to the induction and maintenance of immune homeostasis by dampening immune responses. In fact, stimulation of TLRs might be critically involved in this process and ultimately contribute to preventing development of inflammatory and autoimmune diseases. Sander De Kivit and colleagues present an overview of those aspects, notably outlining the role of gut epithelial TLRs in the induction of immunity and the maintenance of tolerance (1). In addition, the authors highlight the mechanisms through which the gut microbiota regulates intestinal immune responses through interaction with TLRs. The capacity of probiotic microorganisms to modulate immunity via specific TLRs is further discussed in two articles by Julio Villena and Haruki Kitazawa. The authors explore the role of TLR interaction with immunobiotics for the regulation of intestinal inflammation in pigs, with a focus on TLR4 and Lactobacillus jensenii TL2937 (2). Interaction of probiotics with TLR3 to promote beneficial immunity beyond the gut in the respiratory tract is also examined (3). Specifically, inflammatory and immunoregulatory mechanisms conferred by Lactobacillus rhamnosus CRL1505 are presented along with their proposed effects increasing resistance to RSV infection while limiting immunopathology. While TLRs stimulate and regulate immunity against infectious agents, they are also highly pursued targets for therapy of cancer due to their strong ability to activate multiple arms of the immune system, and in particular to stimulate those specific cellular and cytokine responses critical to anti-tumor immunity. However, the immune potentiating, anti-tumor effects of TLR agonists may be restrained by their parallel ability to trigger immune regulation. Dampening of immune responses may enable control of inflammatory damage or immunopathology, but the downside is a limiting effect on efficacy. The impact of this dichotomy on the use of TLR agonists for immunotherapy of cancer is discussed by Hailing Lu in a review article where key immune regulatory facets of TLR agonists are presented, which may impair their efficacy (4). The dual role of TLRs in cancer likely extends beyond the dichotomy between immune stimulation and regulation. Inflammatory processes triggered by TLR engagement may notably constitute a double-edged sword in elimination vs. development of tumors. In that regard, Erin Burns and Nabiha Yusuf contribute two opinion articles discussing TLR targeting for the treatment of cancer. The use of TLR agonists for skin cancer treatment is presented in the context of their impact on skin carcinogenesis, and the question of TLR tolerance is also discussed (5). Similarly, the dual effect of TLR agonists in breast cancer is examined, where TLR stimulation might beneficially activate the immune system but inflammatory processes may also promote tumor development, while TLR-conferred immune regulation may further curb anti-tumor immunity (6). The mechanisms that underlie the immune regulatory properties of TLRs are not well understood. It is possible that the “default” response induced by TLR stimulation may vary between cell types, or depending on the microenvironment/anatomical location of TLR-expressing cells (as exemplified in gut mucosal tissue). Alexandra Zanin-Zhorov and colleagues described an immune regulatory mechanism conferred by TLR expression on T cells, which the authors review herein and discuss in the context of TLR-induced T cell effector functions (7). The role played by endogenous TLR ligands in conferring immune regulatory mechanisms is also presented. Nobuhiro Nakamoto and Takanori Kanai focus on a key organ, the liver, where various types of TLR-expressing cells are faced with continuous exposure to foreign antigens (8). The authors review immune stimulatory and regulatory effects of TLR signaling that coexist in the liver and influence liver health and disease. Elke Gulden and Li Wen concentrate on another organ, the pancreas, where TLR engagement also ultimately controls health and disease (9). Here, the beneficial and detrimental effects of TLR stimulation on type-1 diabetes are discussed, and notably the authors explore how endogenous TLR agonists can confer immune activation vs. regulation of autoreactive T cells. To close on the question of immune regulation conferred by TLRs, Himanshu Singh Chandel and colleagues contribute an opinion article investigating an alternative immune modulatory aspect that may influence the outcome of parasitic infection (10). The authors review mechanisms that underlie cross-talk between TLRs and CD40, and discuss how this interaction may determine the nature of anti-leishmanial immune responses and ultimately parasite elimination.

TLR Agonists for Cancer Immunotherapy: Tipping the Balance between the Immune Stimulatory and Inhibitory Effects

The agonists of toll-like receptors (TLRs) have been actively pursued for their anti-tumor potentials, either as monotherapy or as adjuvants to vaccination or other therapeutic modalities (1). A search on ClinicalTrials.gov using the key words “TLR” and “cancer” returned 34 listings. The idea of using TLR agonist to provide a “danger signal” and break tolerance to tumor antigens has been well embraced by tumor immunologists. However, the promise of TLR agonists-based immunotherapy remains to be realized in the clinic, and only very few TLR agonists have been approved by the FDA. For example, bacillus Calmette–Guerin (BCG) and imiquimod have been approved as standalone therapies, whereas monophosphoryl lipid A (MPL) was approved as a vaccine component. A review of recently published literature on the use of TLR agonists in cancer setting revealed a common mechanism that might have explained the underperformance of TLR agonists as cancer therapeutics: induction of immune suppressive factors that put a break on the TLR agonists-induced inflammation. As shown in Figure ​Figure1,1, TLR agonists have immune stimulatory effects through the induction of costimulatory molecules (CD80, CD86, and CD40) on dendritic cells (DCs) and inflammatory cytokines (TNF-α and IL-12) that polarize Th1 immune response. On the other hand, TLR agonists have immune inhibitory effects as evidenced by the induction of several immune suppressive factors, including IL-10, T regulatory cells (Treg), and PD-L1, all of which could dampen anti-tumor immunity. The following is a brief summary on TLR agonists-induced self-regulatory feedback and the indication for cancer immunotherapy. Figure 1 Schematic diagram showing that PD-1/PD-L1 blockade may enhance TLR-based immunotherapy by tipping the balance between the immune stimulatory and inhibitory effects of TLR agonists. Treatment with TLR agonists in tumor-bearing host not only induces pro-inflammatory ...

Innate immune responses to Proteus mirabilis flagellin in the urinary tract

Flagella are bacterial virulence factors allowing microorganisms to move over surfaces. Flagellin, the structural component of flagella, is sensed by the host via Toll and NOD-like receptors and triggers pro-inflammatory responses. The use of Toll-like receptors agonists to modulate innate immune responses has aroused great interest as an alternative to improve the treatment of diverse infectious diseases. Proteus mirabilis is a Gram negative bacterium that causes urinary tract infections in humans. In the present work we used different approaches to study the ability of P. mirabilis flagellin to induce an innate immune response. We demonstrated that P. mirabilis flagellin has the ability to induce pro-inflammatory chemokines expression in T24 bladder cultures cells and in the mouse bladder after instillation. It was evidenced also that flagellin from different P. mirabilis strains differed in their capacity to induce an innate immune response in the CacoCCL20-Luc system. Also, flagellin elicited inflammation, with recruitment of leukocytes to the bladder epithelium. Flagellin instillation before an experimental P. mirabilis infection showed that the inflammatory response due to flagellin did not help to clear the infection but favored bacterial colonization. Thus, induction of inflammatory response in the bladder did not contribute to P. mirabilis infection neutralization.

Toll-Like Receptor Agonists: Current Status and Future Perspective on their Utility as Adjuvants in Improving Antic Ancer Vaccination Strategies

Toll-like receptor (TLR) agonists possess remarkable properties, particularly with regard to dendritic cell activation, promoting Th1-type cytokine production and optimizing cytotoxic T-cell responses. Preclinical and clinical studies conducted to date show that TLR agonists can improve currently applied anticancer vaccination protocols. Although these have resulted in the US FDA approval of three TLR agonists for use in humans, their abundant application encounters limitations, principally due to dose-limiting toxicity evoking from systemic cytokine production. Here, using selected examples of clinical studies, we provide a concise review regarding the knowledge acquired thus far on the adjuvant use of TLR agonists as cancer vaccine components. We also provide evidence on the exploitation of a novel TLR agonist, prothymosin-α, which enhances the efficacy of tumor-reactive effectors without causing severe adverse effects.

Cancer Vaccination, Will You Have To Pay The Toll?

Cancer vaccines based on patient-derived, autologous immune cells are actively being pursued as a novel strategy to utilize the body’s natural defenses against malignancy. Harnessing the ability of the immune system to fight cancer involves overcoming many obstacles including tumor-specific targeting, overcoming tolerance, and generating effective tumoricidal responses. Co-administration of immune activating adjuvants may hold the key to breaking through several of these barriers. Toll-like receptors (TLR) are pathogen sensors of the innate immune system that activate proinflammatory responses to fight infection and initiate adaptive immune responses. TLRs are increasingly being explored in combination with cancer vaccine strategies since they may have the potential to enhance immunotherapy by promoting tumor-specific immunity. This review will focus on recent basic and clinical research on the use of TLR agonists in cancer therapy.

Toll-Like Receptors as Biomarkers of Gastric Carcinogenesis: Implications for Diagnosis, Prognosis and Treatment

Toll-like receptors (TLR) are essential for Helicobacter pylori (Hp) recognition and subsequent innate and adaptive immunity responses. TLR2 appears to be the receptor responsible for most of the immunologic reaction against Hp infection. However, TLR4, TLR9 and eventually TLR5 may also have a synergic effect with TLR2 against Hp. It has been shown that gastric Hp infection increases TLR expression in the gastric mucosa. Moreover, recent studies have shown that human gastric carcinogenesis is associated not only with increased expression of TLR but also with decreased expression of their inhibitors such as Toll-Interacting Protein (TOLLIP) and peroxisome proliferator-activated receptor (PPAR)-g. Indeed, gastric dysplasia and adenocarcinoma are associated with high expression levels of TLR and low levels of TOLLIP and PPAR-g, suggesting increased activation of these receptors throughout human gastric carcinogenesis. In this article we discuss how these novels findings could be used not only for the diagnosis and prognosis of gastric lesions associated with Hp infection but also for their treatment. Specifically, we discuss the potential use of TLR agonists in addition to antibiotics to improve eradication rates of Hp and of TLR antagonists to slow the progression of gastric preneoplastic lesions. We also discuss the potential value of TLR signalling blockers and quantification of tumoral TLR expression, respectively, in the treatment and prognosis of gastric cancer. In conclusion, TLRs can be an important link between Hp and the sequence of gastric carcinogenesis and they can be used as biomarkers of gastric carcinogenesis. In this article, future lines of investigation related with these novel scientific findings are proposed and discussed.

Toll-Like Receptors in Normal and Malignant Human Bladders

Toll-like receptors have a major role in the innate immune response. They are expressed by immune cells and some epithelial cells. To study the role of urothelial cells in the intrabladder innate immune response we analyzed toll-like receptor expression and functionality in normal and malignant urothelial cells. Toll-like receptor 1 to 10 mRNA expression was analyzed using reverse transcriptase-polymerase chain reaction in 4 primary cultures of normal urothelium and 15 bladder cancer cell lines. Immunohistochemistry was used to detect toll-like receptor expression in 11 normal urothelial samples and 26 bladder tumors. Proinflammatory cytokine secretion by toll-like receptor agonist or bacillus Calmette-Guérin treated cultured cells was assessed by enzyme-linked immunosorbent assay. Mitogen-activated protein kinase phosphorylation was analyzed by Western blot and nuclear factor-κB localization was assessed by confocal microscopy. Expression of most toll-like receptor mRNA was detected in cultured normal or tumor urothelial cells. Expression of toll-like receptors 2 to 4, 5, 7 and 9 protein was detected in all normal urothelial samples and most nonmuscle invasive tumors, although its intensity was decreased in the latter. Expression was markedly decreased in muscle invasive tumors. Treatment with toll-like receptor 2 and 3 agonists showed the strongest inflammatory response in 2 primary cultures of normal urothelial cells and 3 bladder cancer cell lines. Toll-like receptor 2 and 3 functionality was confirmed by the nuclear translocation of nuclear factor-κB and the induction of phosphorylated c-Jun N-terminal kinase mitogen-activated protein-kinase. Toll-like receptors are expressed in normal urothelium and nonmuscle invasive bladder tumors. In cultured urothelial cells agonist inducible toll-like receptor 2 or constitutively expressed toll-like receptor 3 is functional. These data suggest the potential use of toll-like receptor agonists for antitumor immunotherapy of nonmuscle invasive tumors.

Insights into the role of Toll-like receptors in modulation of T cell responses

The innate immune receptors, such as Toll-like receptors (TLRs), are intimately involved in the early sensing of invading microorganisms or their structural components. Engagement of TLRs with their ligands results in activation of several downstream intracellular pathways leading to activation of innate and adaptive immune system cells. It was initially thought that TLRs are primarily expressed by antigen-presenting cells (APCs), such as macrophages and dendritic cells, and that interactions between microbial ligands and TLRs in these cells will indirectly result in activation of cells of the adaptive immune system, especially T cells. However, it has now become evident that TLRs are also expressed by various T cell subsets, such as conventional αβT cells, regulatory T cells, and γδT cells as well as natural killer T cells. Importantly, it appears that at least in some of these T cell subsets, TLRs are functionally active, because stimulation of these cells with TLR agonists in the absence of APCs results in exertion of effector or regulatory functions of T cells. The present review attempts to summarize the recent findings related to TLR expression in different T cell subsets and the direct role of TLRs in the induction and regulation of T cell responses, including those responses that occur at mucosal surfaces. In addition, the potential use of TLR agonists for steering T cell responses as a prophylactic or therapeutic strategy in the context of infectious, allergic or autoimmune diseases is explored.